Langer Stephen J, Ghafoori A Paiman, Byrd Marshall, Leinwand Leslie
Department of Molecular, Cellular and Developmental Biology, CB347, University of Colorado, Boulder, CO 80309-0347, USA.
Nucleic Acids Res. 2002 Jul 15;30(14):3067-77. doi: 10.1093/nar/gkf421.
The ability of the Cre/lox system to make precise genomic modifications is a tremendous accomplishment. However, recombination between cis-linked heterospecific lox sites limits the use of Cre- mediated exchange of DNA to systems where genetic selection can be applied. To circumvent this problem we carried out a genetic screen designed to identify novel mutant spacer-containing lox sites displaying enhanced incompatibility with the canonical loxP site. One of the mutant sites recovered appears to be completely stable in HEK293 cells constitutively expressing Cre recombinase and supports recombinase-mediated cassette exchange (RMCE) in bacteria and mammalian cell culture. By preventing undesirable recombination, these novel lox sites could improve the efficiency of in vivo gene transfer.
Cre/lox系统进行精确基因组修饰的能力是一项巨大成就。然而，顺式连接的异源lox位点之间的重组限制了Cre介导的DNA交换在可应用遗传选择的系统中的使用。为了规避这个问题，我们进行了一项遗传筛选，旨在鉴定与标准loxP位点显示出增强不相容性的新型含突变间隔区的lox位点。回收的一个突变位点在组成型表达Cre重组酶的HEK293细胞中似乎完全稳定，并支持细菌和哺乳动物细胞培养中的重组酶介导的盒式交换（RMCE）。通过防止不期望的重组，这些新型lox位点可以提高体内基因转移的效率。
